The effect of taurine supplementation on the renin–angiotensin–aldosterone system of dogs with congestive heart failure

The role of taurine in the treatment of congestive heart failure (CHF) in dogs without systemic deficiency is unexplored. Taurine might have beneficial cardiac effects aside from deficit replacement. We hypothesized that oral taurine supplementation administered to dogs with naturally-occurring CHF would suppress the renin-angiotensin aldosterone system (RAAS). Oral taurine was administered to 14 dogs with stable CHF. Serum biochemical variables, blood taurine concentrations, and comprehensive analysis of RAAS variables were compared before and 2 weeks after taurine supplementation added to background furosemide and pimobendan therapy for CHF. Whole blood taurine concentrations increased after supplementation (median 408 nMol/mL, range 248–608 before and median 493 nMol/mL, range 396–690 after; P = .006). Aldosterone to angiotensin II ratio (AA2) was significantly decreased after taurine supplementation (median 1.00, range 0.03–7.05 before and median 0.65, range 0.01–3.63 after; P = .009), but no other RAAS components significantly differed between timepoints. A subset of dogs showed marked decreases in RAAS metabolites after supplementation and these dogs were more likely to have been recently hospitalized for CHF treatment than dogs that did not show marked decreases in classical RAAS metabolites. Overall, taurine only lowered AA2 in this group of dogs, however, response heterogeneity was noted, with some dogs showing RAAS suppression.

The only RAAS variable that showed a statistically significant change was AA2, which decreased between visits (P = 0.009). Most other RAAS metabolites decreased between timepoints but the differences did not reach statistical significance (Table 1 and Fig. 1). Seven dogs were hospitalized for the treatment of CHF < 1 week before enrollment and 7 dogs were hospitalized for the treatment of CHF > 1 week before enrollment. Figure 2 shows the individual dog data for angiotensin I, angiotensin II and aldosterone color-coded according to whether dogs were recently hospitalized for the treatment of CHF. All but 1-2 of the highest individual values for angiotensin I, angiotensin II, and aldosterone at V1 were from dogs that were hospitalized within 1 week. Five dogs were classified as RAAS responders and 9 as non-responders. Responders were not more likely than non-responders to be ACE variant positive (P = 0.58) but were more likely to have been discharged from the hospital within a week of enrollment (P = 0.02).
The activity of ACE was significantly lower in ACE variant positive dogs compared to ACE wildtype dogs (P = 0.005) but the ACE-S was not different between genotype groups (P = 0.35). The activity of ACE2 and NEP were not different between genotype groups (P > 0.35).

Discussion
The results of this study were not supportive of a RAAS suppressive effect of oral taurine supplementation for this group of dogs with naturally occurring CHF secondary to mitral valve degeneration. Nearly all the RAAS metabolites decreased, but the only variable that was statistically significantly different between visits was AA2, which reflects the relationship between aldosterone and angiotensin II and is typically viewed as an indicator of adrenal responsiveness to angiotensin II. The AA2 ratio was significantly lower at V2 because a larger decrease in aldosterone occurred compared to angiotensin II. The decrease in aldosterone between visits was notable, but did not reach statistical significance. The clinical significance of this is unknown, but it is possible that nonangiotensin II sources of aldosterone stimulation (e.g. direct beta stimulation) were suppressed by taurine 26  www.nature.com/scientificreports/ Despite the lack of significant differences in RAAS variables after 2 weeks of oral taurine supplementation, a subset of dogs showed decreases in classical RAAS metabolites that were greater than expected for temporal variability, suggesting response heterogeneity within this group of dogs. The decreases in classical metabolite in this subset of dogs were not consistent with an ACE-inhibitory effect, but were global and characterized by decreases in angiotensin I, angiotensin II, and aldosterone which could be a result of adrenergic antagonism as shown in other studies 19,27 . An ACE-inhibitory effect would have been characterized by decreases in angiotensin II and aldosterone and an increase in angiotensin I. Supportive of this was the lack of observable effect of the ACE variant on whether dogs showed decreases in classical RAAS metabolites.
Dogs showing RAAS suppression were more likely to have been recently hospitalized for the treatment of CHF, and these dogs had some of the highest baseline values for classical RAAS metabolites. Dogs with naturally occurring CHF were selected for this study to provide a background of intense RAAS activation due to the presence of advanced heart disease, administration of diuretics, and absence of RAAS-suppressive medications such as ACE-inhibitors, angiotensin-receptor blockers, or mineralocorticoid antagonists. This finding suggests that recent hospitalization might be a cause of even greater RAAS activation for dogs with advanced heart disease and CHF. Contributing factors could include stress, the presence of active CHF, and administration of intravenous diuretic therapy with accompanying volume and electrolyte depletion. The higher body weight at V1 compared to V2 is unexplained, but does not support volume depletion at the initial visit relative to the recheck. One study found that some people with CHF had greater degrees of RAAS activation than other people and this was not related to whether they received low or high doses of diuretic in the hospital 28 . The degree of RAAS activation in our study at V1 was similar to that produced in healthy dogs with the administration of loop diuretics by different routes 29,30 . Additionally, the change in clinical variables (e.g. body weight, heart rate, blood pressure) and biochemical variables reflecting volume status (e.g. albumin, blood urea nitrogen, creatinine) between visits, did not support the presence of significant volume depletion at V1 that was corrected at V2. The increase in serum sodium could be a result of the transition from intravenous to oral diuretic therapy or might be a result of the Table 1. Comparison of clinical, biochemical, and renin-angiotensin aldosterone system variables before (V1) and after (V2) oral taurine supplementation. The data are reported as median (range). P values are from Wilcoxon matched-pairs signed rank test and bold indicates a statistically significant difference between visits. V1; visit 1, V2; visit 2, ACE; angiotensin-converting enzyme, NEP; neutral endopeptidase, AA2; aldosterone to angiotensin II ratio, PRA-S; plasma renin activity surrogate, ACE-S; angiotensin-converting enzyme surrogate, ALT-S; alternative RAAS pathway surrogate. www.nature.com/scientificreports/ observed RAAS suppression as has been noted in people 28 . Regardless of the cause, higher RAAS activation in some dogs at V1 might have contributed to the notable decline in classical RAAS metabolites after taurine supplementation. This provides insight into the mechanism of taurine benefit through global RAAS suppression and similar to studies in hypertensive rats, suggests that benefit might be best realized in patients with the most baseline RAAS activation 27 . Consistent with previous studies of the known ACE variant in dogs, we found that ACE activity was significantly lower in ACE gene variant positive dogs compared to ACE gene wildtype dogs 31 . The lack of difference in ACE-S (a proposed surrogate for ACE activity) between ACE variant and wildtype dogs has also been previously reported 32,33 . Because ACE-S is simply the ratio of angiotensin II to angiotensin I, it appears not to reflect true ACE activity in dogs with the ACE variant because other non-ACE enzymes (e.g. chymase) can form angiotensin II when ACE activity is decreased. Our results did not support an ACE-inhibitory effect even in the subset of dogs that appeared to respond, and so it is not surprising that the ACE variant did not affect the results. Other RAAS polymorphisms could be present in dogs with heart disease.
The activity of ACE2 has not been previous reported in dogs with CHF. The ACE2 activity for this group of CHF dogs was notably higher than reported for both healthy people and people with CHF 34 but was not markedly different from healthy dogs 33 . This enzyme is a marker for disease severity in people with heart disease. More work is needed to understand ACE2 in naturally occurring heart disease in dogs but our data supports

V1
V1 V1 V2 V2 V2 Figure 2. Angiotensin I, angiotensin II, and aldosterone values are shown for each dog at each time point with connecting lines to indicate the same dog. Dots and lines colored red indicate dogs that were hospitalized for treatment of congestive heart failure within 1 week of enrollment. Dots and lines colored black indicate dogs that were hospitalized for treatment of congestive heart failure more than 1 week before enrollment. V1; visit 1 (before taurine supplementation), V2; visit 2 (2 weeks after taurine supplementation). www.nature.com/scientificreports/ notable species differences. Ours is also the first study to report NEP activity in dogs although we did not find a difference between visits for this enzyme. This study has several limitations. Although a priori power analysis indicated a minimum of 12 dogs was needed to show group differences in angiotensin II, our study can still be considered small. The power to detect group differences could have been impacted by unknown factors introducing important variability, including a more advanced stage of heart disease for the included dogs compared to dogs used for sample size calculation. Recent hospitalization for CHF treatment appeared to influence the decrease in classical RAAS activation for a subset of dogs in our study, even though the overall degree of RAAS activation for the group at V1 was similar to previous reports of dogs receiving oral diuretics 29,30 . The use of dogs as their own control, therefore is a weakness of this study (even though week-to-week variability for these metabolites has been reported 35 ), whereas the use of a group receiving placebo would have controlled for confounders such as recent hospitalization. The decreases in most RAAS metabolites did not reach statistical significance between timepoints; while this might indicate a true finding, it could be due to low power resulting from high measurement variability. Evaluation of the effect of taurine in dogs that are naturally RAAS activated and not receiving RAAS inhibiting medications was a strength of this study. Additionally, we documented an increase in whole blood taurine concentration that was well above the reference range after supplementation, although indirect benefits of taurine supplementation might take longer than 2 weeks to be realized (e.g. indirect decreases in RAAS through improved myocardial function).

Conclusions
In conclusion, although we did not find a conclusive RAAS suppressive effect of taurine supplementation in this group of dogs with naturally occurring CHF treated with diuretic therapy and withheld from RAAS suppressive medications, AA2 decreased significantly after supplementation as a result of a greater decrease in aldosterone compared to angiotensin II. Additionally, there was response heterogeneity within this group of dogs, with some showing marked decreases in RAAS metabolite values after supplementation. Other mechanisms through which taurine might benefit dogs with heart disease who are not taurine deficient should be explored in future studies.

Methods
This prospective study was approved by the Institutional Animal Care and Use Committee at the University of Florida College of Veterinary Medicine (#MS1-201910865 and 202200000396) and the authors complied with the ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations. All dog owners provided informed consent.
Inclusion criteria. Dogs being treated for naturally occurring CHF secondary to degenerative mitral valve disease (American College of Veterinary Internal Medicine Stage C) 3 were included if they were stable on oral furosemide and pimobendan and did not have active radiographic or clinical signs of uncontrolled CHF. Dogs with the underlying condition of degenerative mitral valve disease were targeted for enrollment instead dogs with dilated cardiomyopathy due to the higher frequency of degenerative mitral valve disease in the canine population. Both conditions lead to the same end result of CHF with common pathophysiology. Dogs were eligible for enrollment after being treated in the hospital for left-sided CHF once they were transitioned to oral medications. A body weight greater than 4 kg was required to facilitate blood sampling.
Exclusion criteria. Dogs that received RAAS-suppressive therapies (e.g. ACE-inhibitors, angiotensin receptor blockers, spironolactone) or taurine supplementation within the previous 2 weeks were excluded. Dogs with right-sided CHF were excluded.
Procedures. Timeline. Buccal swab and blood (8 mLs) collected by peripheral venipuncture were obtained at V1 for biochemical analysis, RAAS analysis, taurine concentrations and ACE genotyping. Taurine supplementation (Now® or PetAg® brands) was started after V1 (target daily dosage 60 mg/kg/day divided into 2 doses 12 h apart, dose was rounded up to fit tablet or capsule sizes) in addition to current cardiac medications. Blood samples were collected again 2 weeks later (V2) for biochemical analysis, RAAS analysis, and taurine concentrations.
Collection of clinical data. Breed, age, sex, body weight, systolic blood pressure (by Doppler), heart rate, medication dosages, and time (< or > 1 week) from last hospitalization for CHF were recorded for each dog.
Serum biochemical analysis: Five mLs of blood were placed into additive-free tubes and centrifuged at 3000 rpm for 10 min. Serum was removed and placed into 2 aliquots. One aliquot was submitted to the University of Florida Clinical Pathology Laboratory for biochemical analysis (blood urea nitrogen, creatinine, albumin, sodium chloride, potassium, calcium, phosphorous, anion gap). The 2nd aliquot was stored at −80 °C until batch analysis for RAAS analysis.
Blood taurine concentrations. Three mLs of blood were placed into 2 lithium heparin tubes, one of which was centrifuged at 3000 rpm for 10 min for plasma removal. The plasma sample and the whole blood sample were shipped on dry ice to the University of California, Davis, Amino Acid Laboratory (Davis, CA) for measurement of whole blood and plasma taurine concentrations.
Genotyping. The buccal swab was used to genotype dogs for the known ACE variant which can affect baseline ACE activity and aldosterone breakthrough 32  www.nature.com/scientificreports/ dna-testi ng/). Nucleotide sequences were visually evaluated for sequence quality and aligned using sequence analysis software to determine the presence or absence of the ACE gene polymorphism.
Equilibrium analysis of the RAAS. This study uses the methods described in previous studies and this methods description partly reproduces their wording [32][33][34] . The equilibrium concentrations of 6 different angiotensin peptide metabolites (angiotensin I, angiotensin II, angiotensin III, angiotensin IV, angiotensin 1-5, angiotensin 1-7) and aldosterone were quantified by liquid chromatography-mass spectrometry/mass-spectroscopy (LC-MS/MS), performed by a service provider laboratory (Attoquant Diagnostics, Vienna, Austria), using previously validated and described methods 14 . Analyte concentrations were reported in pmol/L and the lower limit of quantification was reported for each metabolite. The activities of ACE and ACE2 were measured with a kinetics approach using spiked substrate 33,36 . Activity of ACE was determined by measuring the formation of angiotensin II after addition of angiotensin I in the presence and absence of an ACE inhibitor and a chymase inhibitor to determine the ACE-inhibitor sensitive fraction of angiotensin II generation. Activity of ACE2 was determined by measuring the formation of angiotensin 1-7 after the addition of angiotensin II in the presence and absence of an ACE2 inhibitor using recombinant human ACE2 as a reference standard. Activity of neutral endopeptidase (NEP) was determined by measuring the formation of angiotensin 1-7 after addition of angiotensin I in the presence and absence of a NEP inhibitor. The ratio of angiotensin II to angiotensin I was calculated as a marker for ACE activity (ACE-S) 32 . Angiotensin I and angiotensin II were summed as a marker for plasma renin activity (PRA-S) 37 . The ratio of aldosterone to angiotensin II (AA2) was calculated as a unitless indicator of adrenal responsiveness to angiotensin II stimulation of aldosterone release 32 . The sum of angiotensins 1-7 and 1-5 divided by the sum of angiotensins I, II, 1-7, and 1-5, was calculated as a unitless marker of renin-independent alternative RAAS activation (ALT-S) 33 . The ratio of angiotensin 1-7/angiotensin I was calculated to account for overall RAAS activity to aid interpretation of the balance of angiotensin 1-7 formation and breakdown 33 .
Statistical analysis. A priori power analysis showed a minimum of 12 dogs would be needed to show a significant decrease in angiotensin II concentrations between V1 and V2 with a power of 80% and significance level of 5%, using the decrease in angiotensin II after enalapril administration in a previous study of dogs with preclinical mitral valve disease for this calculation (mean 43 pM ± standard deviation 47 pM) 32 . Clinical data, biochemical data, RAAS data, and taurine data were assessed for normality using Shapiro-Wilk test, and median (range) were reported for each visit (V1, V2). Variables were compared between V1 and V2 using Wilcoxon matchedpairs signed rank test. Individual dogs were considered RAAS responsive if they showed a decrease in at least 3 classical RAAS components (angiotensin I, angiotensin II, angiotensin III, angiotensin IV, or aldosterone) of > 55% to account for temporal variability at V2 35 . Fisher's exact test was used to assess if ACE genotype or time from CHF episode (< 1 week, > 1 week) was associated with being RAAS responsive. The activity of ACE, ACE2, and NEP were compared between ACE genotypes using Mann-Whitney test. Metabolite concentrations below the lower limit of quantification were entered as half the lower limit of quantification for statistical comparisons. Commercially available statistical software was used for analysis (GraphPad Prism 9). P < 0.05 was considered statistically significant.
Institutional animal care and use committee (IACUC) or other approval declaration. This study was approved by the Institutional Care and Use Committee at the University of Florida, College of Veterinary Medicine (#MS1-201910865 and 202200000396).
Human ethics approval declaration. Authors declare human ethics approval was not needed for this study.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information file).